This invention relates to separation systems and their components and more particularly to disposable columns that include monolithic permeable polymeric materials as packing.
It is known to use monolithic macroporous materials for packing in chromatographic columns. The prior art monolithic columns have several disadvantages, such as, (1) they are relatively expensive compared to some disposable columns; (2) they have little more or less resolution and speed than the conventional columns packed with either silica beads or polymer beads, particularly with respect to separation of large molecules; (3) the wide pore size distribution that results from stacking of the irregular particles with various shapes and sizes lowers the column efficiency; (4) the non-homogeneity of the pore sizes resulting from the non-homogeneity of the particle sizes and shapes in the above materials contribute heavily to the zone spreading; (5) the large amount of micropores in the prior art monolithic packing also contributes greatly to the zone spreading; and (6) shrinkage of the material used in the columns reduces the efficiency of the columns. These problems limit their use in high resolution chromatography.
Inexpensive disposable chromatographic columns are known from U.S. Pat. No. 6,565,745, entitled DISPOSABLE CHROMATOGRAPHIC COLUMNS. These disposable columns are manufactured of inexpensive plastics and designed to be easily assembled by filling the body of the column with the desired packing and then welding the open end or ends closed. The prior art chromatographic column generally used has the disadvantage of having low resolution and speed.
Prior art European Patent 1,188,736 describes a method of making porous poly(ethylene glycol methacrylate-co-ethylene glycol dimethacrylate) by in situ copolymerization of a monomer, a crosslinking agent, a porogenic solvent and an initiator inside a polytetrafuoroethylene tube sealed at one end and open at the other end. The resulting column was used for gas-liquid chromatography. This prior art approach has the disadvantage of not resulting in materials having the characteristics desirable for the practical uses in liquid chromatography.
U.S. Pat. Nos. 5,334,310; 5,453,185 and 5,728,457 each disclose a method of making macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) polystyrene in situ within sealed columns. This method extends the methods described in both the European patent 1,188,736 and U.S. Pat. Nos. 2,889,632; 4,923,610 and 4,952,349 for preparing liquid chromatography columns for the separation of proteins. U.S. Pat. Nos. 5,334,310; 5,453,185 and 5,728,457 profess the intention of improving the column efficiency by removing the interstitial volume of conventional packed columns having beads. The plugs formed according to these patents have a separation-effective opening size distribution that is controlled by the type and amount of porogens, monomers and polymerization temperature. The macroporous polymers consist of interconnected aggregates of particles of various sizes which form large pore channels between the aggregates for the transport of the mobile phase. Among the aggregates or clusters there exist small pores for separations. The small particles are formed from tightly packed extremely small particles ca 100-300 nanometers.
The materials made in accordance with these patents have a disadvantage in that the micropores within or between these particles physically trap the sample molecules and degrade the separation. Although these patents claim that there are no interstitial spaces in the monolithic media as in the packed bed with beads, the large channels between the aggregates and interconnected particles actually cause the same problem as the interstitial spaces between the beads in conventional packed columns with beads. The large channels formed from various sizes of aggregates or clusters are inhomogeneous and provide random interstitial spaces, even with narrow particle size distribution. Because of the random interstitial spaces the column efficiency is poor.
U.S. Pat. Nos. 5,334,310; 5,453,185 and 5,728,457 disclose the preparation of the separation media inside a column with cross section area from square micrometers to square meters. The processes disclosed in these patents have some disadvantages. Some of the disadvantages were disclosed by the inventors named in those patents in 1997 in Chemistry of Materials, 1997, 9, 1898.
One significant disadvantage is that larger diameter (26 mm I.D.) columns prepared from the above patented process have a separation-effective opening size distribution that is too irregular to be effective in chromatography separation. The irregular distribution of the sizes of the separation-effective openings is caused by the detrimental effect of polymerization exotherm, the heat isolating effect of the polymer, the inability of heat transfer, autoaccelerated decomposition of the initiator and concomitant rapid release of nitrogen by using azobisisobutyronitrile as initiator in a mold with 26 mm diameter. It has been found that the temperature increase and differential across the column created by the polymerization exotherm and heat transfer difficulties results in accelerated polymerization in large diameter molds such as for example molds having a diameter of more than 15 mm and in a temperature gradient between the center of the column and the exterior wall of the column which results in inhomogeneous pore structure. It was suggested in this article that the problem might be reduced by slow addition of polymerization mixture. This helps to solve the problem partly but does not solve the problem completely. There is still a temperature gradient for the larger diameter columns, which results in a lack of homogeneity in the separation-effective opening size distribution.
This problem was also verified by theoretical calculations in the publication of Analytical Chemistry, 2000, 72, 5693. This author proposes a modular approach by stacking thin cylinders to construct large diameter columns for radial flow chromatography. However, sealing between the discs to form a continuous plug is difficult and time consuming.
U.S. Pat. Nos. 5,334,310; 5,453,185 and 5,728,457 disclose the material of weak anion exchange and reversed phase columns. The weak anion exchanger prepared had low resolution, low capacity, low rigidity, slow separation and very poor reproducibility. The reversed phase media has very little capacity, non-ideal resolution, and very poor reproducibility. It can not be used in mobile phase with high water content such as less than 8% acetonitrile in water due to wall channeling effect resulting from shrinkage of the very hydrophobic media in this very polar mobile phase. This media is also compressed during separation and results in excess void volume in the head of the column. The above patents provide little guidance on how to prepare a weak cation exchanger, strong cation exchanger, strong anion exchanger, normal phase media and hydrophobic interaction media. These media based on membrane, beads or gels are known. However, the known preparation is performed off-line and can not be used for in situ preparation of monolithic columns. The monolithic membranes prepared according to U.S. Pat. Nos. 2,889,632; 4,923,610 and 4,952,349 have low capacity and resolution.
It is known from Peters et al., “Preparation of Large-Diameter ‘Molded’ Porous Polymer Monoliths and the Control of Pore Structure Homogeneity”, Chemistry of Materials v. 9, n7, July 1997 to polymerize the monolithic plug slowly in an effort to avoid temperature gradients. In one embodiment, the temperature is held at a constant low temperature of 55 degrees Centigrade or at 60 degrees Centigrade. In another embodiment, the rate of polymerization is maintained low and exothermal heat reduced by gradually adding monomer and thus limiting the rate of reaction.
These processes have the disadvantage of providing inhomogeneous separation-effective opening size distribution, low stiffness of the polymer plug and separation-effective openings that are impractically large for many liquid chromatography separations. The low temperature results in reduced cross linking and, for some polymers, results in a plug that is not sufficiently stiff.
This publication also describes the process by which the authors obtained information about the internal temperature of the columns during polymerization. That process included the embedding of temperature measuring devices in the column.